Method of treating fibroproliferative disorders

ABSTRACT

Materials and methods for reducing cell proliferation or extracellular matrix production in a mammal are disclosed. The methods comprise administering to a mammal a composition comprising a therapeutically effective amount of a zvegf4 antagonist in combination with a pharmaceutically acceptable delivery vehicle. Exemplary zvegf4 antagonists include anti-zvegf4 antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation, and mitogenically inactive, receptor-binding variants of zvegf4. The materials and methods are useful in the treatment of, inter alia, fibroproliferative disorders of the kidney, liver, and bone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/808,972,filed Mar. 14, 2001, now U.S. Pat. No. 6,630,142, which is incorporatedherein by reference, which claims the benefit of provisional applicationSer. No. 60/235,295, filed Sep. 26, 2000, and which is acontinuation-in-part of application Ser. No. 09/564,595, filed May 3,2000, now U.S. Pat. No. 6,495,668, which claims the benefit ofprovisional applications Ser. No. 60/132,250, filed May 3, 1999, Ser.No. 60/164,463, filed Nov. 10, 1999, and Ser. No. 60/180,169, filed Feb.4, 2000.

BACKGROUND OF THE INVENTION

Fibroproliferative disorders are characterized by the abnormalaccumulation of fibrous tissue (“fibrosis”) that can occur as a part ofthe wound-healing process in damaged tissue. Such tissue damage mayresult from physical injury, inflammation, infection, exposure totoxins, and other causes. The fibroproliferative condition includes botha cell growth component and an extensive phase characterized byextracellular matrix accumulation. Examples of fibroproliferativedisorders include dermal scar formation, keloids, liver fibrosis, lungfibrosis (e.g., silicosis, asbestosis), kidney fibrosis (includingdiabetic nephropathy), and glomerulosclerosis.

A variety of renal diseases can be classified as fibroproliferative.Glomerular (usually mesangial) cell proliferation occurs in many typesof glomerulonephritides in conjunction with increased extracellularmatrix accumulation (Iida et al., Proc. Natl. Acad. Sci. USA88:6560–6564, 1991). For example, mesangial cell proliferation precedesglomerulosclerosis in the remnant kidney model (Floege et al., KidneyInternational 41:297–309, 1992), and experimental overexpression ofgrowth factors such as PDGF-B and TGF-beta in the kidney induces cellproliferation, matrix accumulation, and glomerulosclerosis (Isaka etal., J. Clin. Invest. 92:2597–2601, 1993; Cybulsky, Curr. Opin.Nephropathy and Hypert. 9:217–223, 2000).

A number of vascular pathologies result from a combination ofmesenchymal cell proliferation (smooth muscle and fibroblast-like) andextensive accumulation of extracellular matrix components. Such arterywall diseases as arteriosclerotic lesions, arteritis of various origins,and the vascular re-stenotic lesions that frequently follow angioplasty(Riessen et al., Am. Heart J. 135:357–364, 1998; Plenz et al.,Arterioscler. Thromb. Vasc. Biol. 17:2489–2499, 1997; McCaffrey,Cytokine Growth Factor Rev. 11:103–114, 2000) are consideredfibroproliferative. Other fibroproliferative responses include thefiborproliferative responses that occur in organs following transplant(e.g., heart transplants), at sites of vascular anastamosis, and atareas around catheter placements (e.g., arterio-venous shunts used fordialysis).

Bone formation, both physiologic and pathologic, can be described as theinterplay between bone formation that results from proliferation ofosteoblasts and production by them of extracellular matrix, and thereplication of osteoclasts and their modulation of this matrix. Diseaseswhere there is aberrant and ectopic bone formation, such as thatoccurring with prostate tumor metastases to the axial skeleton, arecommonly characterized by active proliferation of the major cell typesparticipating in bone formation as well as by elaboration by them of acomplex bone matrix. These diseases can therefore be viewed asfibroproliferative.

Pulmonary fibrosis is a major cause of morbidity and mortality.Pulmonary fibrosis is associated with the use of high-doseantineoplastic agents (e.g., bleomycin) in chemotherapy and with bonemarrow transplantation for cancer treatment. The development of lungdisease is the major dose-limiting side effect of bleomycin. See, Tranet al., J. Clin. Invest. 99:608–617, 1997. Idiopathic pulmonary fibrosis(IPF) is another lung fibrotic disease characterized by afibroproliferative response. Various factors, including aspiration andexposure to environmental pollutants may result in IPF (Egan, The Lancet354:1839–1840, 1999). The standard treatment for IPF is oralglucocorticoids. However, lung function improves in less than 30 percentof patients who receive this treatment, and, regardless of treatment,the median survival is four to five years after the onset of symptoms.The proliferation of fibroblasts and the accumulation of interstitialcollagens are the hallmarks of progressive organ fibrosis, however thebiochemical mechanism of induction of lung fibrosis remains unclear(Ziesche et al., New Eng. J. Med. 341:1264–1269, 1999; Kuwano et al., J.Clin Invest. 104:13–19, 1999). Pulmonary hypertension results from avariety of initiating stimuli. Its progression is associated withpulmonary vascular sclerosis, which includes abnormal endothelialmorphology and function, muscularization of normally nonmuscularperipheral arteries related to differentiation of pericytes, and medialhypertrophy and neointimal formation in muscular arteries as aconsequence of hypertrophy, proliferation, and migration of residentsmooth muscle cells and increased production of extracellular matrixcomponents. These components include collagen, elastin, fibronectin, andtenascin-C. This fibroproliferative response can progress tolife-threatening pulmonary arterial obstructive disease (Cowan et al.,J. Clin. Invest. 105:21–34, 2000).

Liver (hepatic) fibrosis occurs as a part of the wound-healing responseto chronic liver injury. Fibrosis occurs as a complication ofhaemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viralhepatitis, bile duct obstruction, toxin exposure, and matabolicdisorders. This formation of scar tissue is believed to represent anattempt by the body to encapsulate the injured tissue. Liver fibrosis ischaracterized by the accumulation of extracellular matrix that can bedistinguished qualitatively from that in normal liver. Left unchecked,hepatic fibrosis progresses to cirrhosis (defined by the presence ofencapsulated nodules), liver failure, and death.

In recent years there have been significant advances in theunderstanding of the cellular and biochemical mechanisms underlyingliver fibrosis (reviewed by Li and Friedman, J. Gastroenterol. Hepatol.14:618–633, 1999). Stellate (Ito) cells are believed to be a majorsource of extracellular matrix in the liver. Stellate cells respond to avariety of cytokines present in the liver, some of which they alsoproduce (Friedman, Seminars in Liver Disease 19:129–140, 1999).

As summarized by Li and Friedman (ibid.), actual and proposedtherapeutic strategies for liver fibrosis include removal of theunderlying cause (e.g., toxin or infectious agent), suppression ofinflammation (using, e.g., corticosteroids, IL-1 receptor antagonists,or other agents), down-regulation of stellate cell activation (using,e.g., gamma interferon or antioxidants), promotion of matrixdegradation, or promotion of stellate cell apoptosis. Despite recentprogress, many of these strategies are still in the experimental stage,and existing therapies are aimed at suppressing inflammation rather thanaddressing the underlying biochemical processes. Thus, there remains aneed in the art for materials and methods for treatingfibroproliferative disorders, including liver fibrosis.

DESCRIPTION OF THE INVENTION

Within one aspect of the present invention there is provided a method ofreducing proliferation of or extracellular matrix production by a cellin a mammal comprising administering to the mammal a compositioncomprising a therapeutically effective amount of a zvegf4 antagonist incombination with a pharmaceutically acceptable delivery vehicle, whereinthe zvegf4 antagonist is selected from the group consisting ofanti-zvegf4 antibodies, inhibitory polynucleotides, inhibitors of zvegf4activation, and mitogenically inactive, receptor-binding variants ofzvegf4. Within certain embodiments of the invention the proliferation ofmesangial, epithelial, endothelial, smooth muscle, fibroblast,osteoblast, osteoclast, neuronal, stromal, stellate, or interstitialcells is reduced. Within another embodiment of the invention theproliferation of tumor cells, such as prostate tumor cells, is reduced.Within another embodiment of the invention extracellular matrixproduction is reduced. Within other embodiments of the invention themammal is suffering from a fibroproliferative disorder of the kidney,liver, or bone.

Within a related aspect of the invention there is provided a method ofreducing proliferation of or extracellular matrix production by a cellin a mammal, wherein the cell is an epithelial, endothelial, smoothmuscle, fibroblast, osteoblast, neuronal, or stellate cell, the methodcomprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation,and mitogenically inactive, receptor-binding variants of zvegf4.

Within a further aspect of the invention there is provided a method ofreducing proliferation of or extracellular matrix production by prostatetumor cells in a mammal, the method comprising administering to themammal a composition comprising a therapeutically effective amount of azvegf4 antagonist in combination with a pharmaceutically acceptabledelivery vehicle, wherein the zvegf4 antagonist is selected from thegroup consisting of anti-zvegf4 antibodies, inhibitory polynucleotides,inhibitors of zvegf4 activation, and mitogenically inactive,receptor-binding variants of zvegf4.

Within another aspect of the invention there is provided a method ofreducing metastasis of prostate cancer cells to bone in a mammal, themethod comprising administering to the mammal a composition comprising atherapeutically effective amount of a zvegf4 antagonist in combinationwith a pharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitory polynucleotides, inhibitors of zvegf4 activation,and mitogenically inactive, receptor-binding variants of zvegf4.

Within a further aspect of the invention there is provided a method oftreating a fibroproliferative disorder in a mammal comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of a zvegf4 antagonist in combination with apharmaceutically acceptable delivery vehicle, wherein the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies, inhibitors of zvegf4 activation, mitogenically inactivereceptor-binding zvegf4 variant polypeptides, and inhibitorypolynucleotides. Within certain embodiments of the invention thefibroproliferative disorder is a fibroproliferative disorder of liver,kidney, or bone.

Within an additional aspect of the invention there is provided a methodof reducing stellate cell activation in a mammal comprisingadministering to the mammal a composition comprising a zvegf4 antagonistin combination with a pharmaceutically acceptable delivery vehicle,wherein the zvegf4 antagonist is selected from the group consisting ofanti-zvegf4 antibodies, mitogenically inactive receptor-binding zvegf4variant polypeptides, and inhibitory polynucleotides, in an amountsufficient to reduce stellate cell activation.

Within certain embodiments of the above-disclosed methods, the zvegf4antagonist is selected from the group consisting of anti-zvegf4antibodies and inhibitory polynucleotides. Within other embodiments, thezvegf4 antagonist is an anti-zvegf4 antibody. Within additionalembodiments of these methods, the zvegf4 antagonist is administered incombination with an antagonist of a second growth factor.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theaccompanying figure.

FIGS. 1A–1G are a Hopp/Woods hydrophilicity profile of the amino acidsequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the figure bylower case letters.

The term “antagonist” is used herein to denote a compound that reduces abiological activity of another compound. Within the present invention, a“zvegf4 antagonist” is a compound that reduces the receptor-mediatedbiological activity (e.g., mitogenic activity) of zvegf4 on a targetcell. Antagonists may exert their action by competing with zvegf4 forbinding sites on a cell-surface receptor, by binding to zvegf4 andpreventing it from binding to a cell-surface receptor, by otherwiseinterfering with receptor function, by reducing production of zvegf4, orby other means.

“Extracellular matrix” (ECM) is a complex mixture of macromolecules thataccumulates within tissues in close apposition to cell surfaces. ECMcontains secreted macromolecules such as collagens I, III and IV;fibronectin; laminins; and various proteoglycans. These macromoleculescan be organized to provide cohesion to the tissue and can contribute toits structural and mechanical properties. ECM can act as a depositoryfor, and release site of, potent secreted growth factors, and is knownto influence growth, survival and differentiation of the cells itsurrounds. Pathologic ECM accumulation, if unchecked, can restrictaccess of nutrients, growth factors, and other physiologically importantmolecules to cells and can lead to the creation of areas of low livecell density. Over time, this accumulation can result in the inabilityof a tissue to perform its specific metabolic and structural roles, andmay ultimately lead to overt cell and tissue death.

An “inhibitory polynucleotide” is a DNA or RNA molecule that reduces orprevents expression, (transcription or translation) of a second (target)polynucleotide. Inhibitory polynucleotides include antisensepolynucleotides, ribozymes, and external guide sequences. The term“inhibitory polynucleotide” further includes DNA and RNA molecules thatencode the actual inhibitory species, such as DNA molecules that encoderibozymes.

The terms “treat” and “treatment” are used broadly to denote therapeuticand prophylactic interventions that favorably alter a pathologicalstate. Treatments include procedures that moderate or reverse theprogression of, reduce the severity of, prevent, or cure a disease.

The term “zvegf4 protein” is used herein to denote proteins comprisingthe growth factor domain of a zvegf4 polypeptide (e.g., residues 258–370of human zvegf4 (SEQ ID NO:2) or mouse zvegf4 (SEQ ID NO:4)), whereinthe protein is mitogenic for cells expressing cell-surface PDGF α-and/or β-receptor subunit. Zvegf4 has been found to activate the αα, αβ,and ββ isoforms of PDGF receptor. Zvegf4 proteins include homodimers andheterodimers as disclosed below. Using methods known in the art, zvegf4proteins can be prepared in a variety of forms, including glycosylatedor non-glycosylated, pegylated or non-pegylated, with or without aninitial methionine residue, and as fusion proteins as disclosed in moredetail below.

All references cited herein are incorporated by reference in theirentirety.

The present invention provides methods for reducing proliferation of orextracellular matrix production by a cell in a mammal using zvegf4antagonists. The invention further provides methods of treatingfibroproliferative disorders in a mammal using zvegf4 antagonists.Zvegf4 is a multi-domain protein that is structurally related toplatelet-derived growth factor (PDGF) and the vascular endothelialgrowth factors (VEGF). This protein is also referred to as “PDGF-D”(WIPO Publication WO 00/27879).

Structural predictions based on the zvegf4 sequence and its homology toother growth factors suggests that the polypeptide can formhomomultimers or heteromultimers that act on tissues by modulating cellproliferation, migration, differentiation, or metabolism. Experimentalevidence supports these predictions. Zvegf4 heteromultimers may comprisea polypeptide from another member of the PDGF/VEGF family of proteins,including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3/PDGF-C (WO 00/34474),P1GF (Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267–9271, 1991),PDGF-A (Murray et al., U.S. Pat. No. 4,899,919; Heldin et al., U.S. Pat.No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123–129, 1984; Johnssonet al., EMBO J. 3:921–928, 1984).

The zvegf4 polypeptide chain comprises a growth factor domain, a CUBdomain, and an interdomain linking the CUB and growth factor domains.The growth factor domain is characterized by an arrangement of cysteineresidues and beta strands that is characteristic of the “cystine knot”structure of the PDGF family. The CUB domain shows sequence homology toCUB domains in the neuropilins (Takagi et al., Neuron 7:295–307, 1991;Soker et al., Cell 92:735–745, 1998), human bone morphogenetic protein-1(Wozney et al., Science 242:1528–1534, 1988), porcine seminal plasmaprotein and bovine acidic seminal fluid protein (Romero et al., Nat.Struct. Biol. 4:783–788, 1997), and X. laevis tolloid-like protein (Linet al., Dev. Growth Differ. 39:43–51, 1997).

A representative human zvegf4 polypeptide sequence is shown in SEQ IDNO:2, and a representative mouse zvegf4 polypeptide sequence is shown inSEQ ID NO:4. DNAs encoding these polypeptides are shown in SEQ ID NOS: 1and 3, respectively. Analysis of the amino acid sequence shown in SEQ IDNO:2 indicates that residues 1 to 18 form a secretory peptide. The CUBdomain extends from residue 52 to residue 179. A propeptide-likesequence extends from residue 180 to either residue 245, residue 249 orresidue 257, and includes four potential cleavage sites at its carboxylterminus, monobasic sites at residue 245 and residue 249, a dibasic siteat residues 254–255, and a target site for furin or a furin-likeprotease at residues 254–257. Protein produced in a baculovirusexpression system showed cleavage between residues 249 and 250, andincluded longer species with amino termini at residues 19 and 35. Thegrowth factor domain extends from residue 258 to residue 370, and mayinclude additional residues at the N-terminus (e.g., residues 250 to 257or residues 246 to 257). Those skilled in the art will recognize thatdomain boundaries are somewhat imprecise and can be expected to vary byup to ±5 residues from the specified positions. Corresponding domains inmouse and other non-human zvegf4s can be determined by those of ordinaryskill in the art from sequence alignments. Cleavage of full-length humanzvegf4 with plasmin resulted in activation of the zvegf4 polypeptide asdetermined in a cellular assay using a PDGF receptor and luciferasereporter gene. By Western analysis, a band migrating at approximatelythe same size as the growth factor domain was observed in thispreparation.

Signal peptide cleavage is predicted to occur in human zvegf4 afterresidue 18 (±3 residues). Upon comparison of human and mouse zvegf4sequences, alternative signal peptide cleavage sites are predicted afterresidue 23 and/or residue 24. This analysis suggests that the zvegf4polypeptide chain may be cleaved to produce a plurality of monomericspecies, some of which are shown in Table 1. In certain host cells,cleavage after Lys-255 is expected to result in subsequent removal ofresidues 254–255, although polypeptides with a carboxyl terminus atresidue 255 may also be prepared. Cleavage after Lys-257 is expected toresult in subsequent removal of residue 257. These cleavage sites can bemodified to prevent proteolysis and thus provide for the production ofuncleaved zvegf4 polypeptides and multimers comprising them. Actualcleavage patterns are expected to vary among host cells.

TABLE 1 Monomer Residues (SEQ ID NO:2) CUB domain 19–179 24–179 25–17935–179 52–179 CUB domain + interdomain region 19–257 24–257 25–25735–257 52–257 19–255 24–255 25–255 35–255 52–255 19–253 24–253 25–25335–253 52–253 19–249 24–249 25–249 35–249 52–249 19–245 24–245 25–24535–245 52–245 CUB domain + interdomain region + 19–370 growth factordomain 24–370 25–370 35–370 52–370 Growth factor domain 246–370 250–370  258–370  Growth factor domain + 180–370  interdomain region

Zvegf4 can thus be prepared in a variety of multimeric forms comprisinga zvegf4 polypeptide as disclosed above. These zvegf4 polypeptidesinclude zvegf4₁₉₋₃₇₀, zvegf4₅₂₋₃₇₀, zvegf4₂₄₆₋₃₇₀, zvegf4₂₅₀₋₃₇₀, andzvegf4₂₅₈₋₃₇₀. Variants and derivatives of these polypeptides can alsobe prepared as disclosed herein.

Zvegf4 proteins can be prepared as fusion proteins comprising amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, an affinity tag, or a targetting polypeptide. For example, azvegf4 protein can be prepared as a fusion with an affinity tag tofacilitate purification. In principal, any peptide or protein for whichan antibody or other specific binding agent is available can be used asan affinity tag. Affinity tags include, for example, a poly-histidinetract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith andJohnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al.,Proc. Natl. Acad. Sci. USA 82:7952–4, 1985), substance P, FLAG peptide(Hopp et al., Biotechnology 6:1204–1210, 1988), streptavidin bindingpeptide, maltose binding protein (Guan et al., Gene 67:21–30, 1987),cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, orother antigenic epitope or binding domain. Fusion of zvegf4 to, forexample, maltose binding protein or glutatione S transferase can be usedto improve yield in bacterial expression systems. In these instances thenon-zvegf4 portion of the fusion protein ordinarily will be removedprior to use. Separation of the zvegf4 and non-zvegf4 portions of thefusion protein is facilitated by providing a specific cleavage sitebetween the two portions. Such methods are well known in the art. Zvegf4can also be fused to a targetting peptide, such as an antibody(including polyclonal antibodies, monoclonal antibodies, antigen-bindingfragments thereof such as F(ab′)₂ and Fab fragments, single chainantibodies, and the like) or other peptidic moiety that binds to atarget tissue.

Variations can be made in the zvegf4 amino acid sequences shown in SEQID NO:2 and SEQ ID NO:4 to provide inactive, receptor-bindingpolypeptides that act as zvegf4 antagonists. Such variations includeamino acid substitutions, deletions, and insertions. While not wishingto be bound by theory, it is believed that residues within regions273–295 and 307–317 of human zveg4 (SEQ ID NO:2) may be involved inligand-receptor interactions. It is also believed that the CUB domainmay mediate the binding of zvegf4 to certain cell-surface receptors,thereby providing a targeting function for delivery of the growth factordomain. The CUB domain, in the absence of an active growth factordomain, may therefore be useful as a zvegf4 antagonist. The effects ofamino acid sequence changes at specific positions in zvegf4 proteins canbe assessed using procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244, 1081–1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA88:4498–4502, 1991). Multiple amino acid substitutions can be made andtested using known methods of mutagenesis and screening, such as thosedisclosed by Reidhaar-Olson and Sauer (Science 241:53–57, 1988) or Bowieand Sauer (Proc. Natl. Acad. Sci. USA 86:2152–2156, 1989). Other methodsthat can be used include phage display (e.g., Lowman et al., Biochem.30:10832–10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/06204), region-directed mutagenesis (Derbyshire etal., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988), and DNA shuffling(Stemmer, Nature 370:389–391, 1994; Stemmer, Proc. Natl. Acad. Sci. USA91:10747–10751, 1994). The resultant mutant molecules are tested forreceptor binding, mitogenic activity, or other properties (e.g.,stimulation of extracellular matrix production) to identify amino acidresidues that are critical to these functions. Mutagenesis can becombined with high-volume or high-throughput screening methods to detectbiological activity of zvegf4 variant polypeptides, including biologicalactivity in modulating cell proliferation. For example, mitogenesisassays that measure dye incorporation or ³H -thymidine incorporation canbe carried out on large numbers of samples. Competition assays can beemployed to confirm antagonist activity.

Zvegf4 proteins, including full-length proteins, variant proteins(including antagonists), biologically active fragments, and fusionproteins, can be produced in genetically engineered host cells accordingto conventional techniques. Suitable host cells are those cell typesthat can be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells (including cultured cells of multicellular organisms).Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, Green and Wiley and Sons,NY, 1993. In general, a DNA sequence encoding a zvegf4 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers,vectors, and other elements is a matter of routine design within thelevel of ordinary skill in the art. Many such elements are described inthe literature and are available through commercial suppliers. See, forexample, WO 00/34474.

Zvegf4 proteins can comprise non-naturally occurring amino acidresidues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N -methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806–809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145–10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991–19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470–7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards. ProteinSci. 2:395–403, 1993).

Zvegf4 polypeptides or fragments thereof can also be prepared throughchemical synthesis according to methods known in the art, includingexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989.

Zvegf4 proteins are purified by conventional protein purificationmethods, typically by a combination of chromatographic techniques. See,in general, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York, 1994. Proteinscomprising a polyhistidine affinity tag (typically about 6 histidineresidues) are purified by affinity chromatography on a nickel chelateresin. See, for example, Houchuli et al., Bio/Technol. 6: 1321–1325,1988. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952–4,1985. Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art. Zvegf4 growth factor domainprotein can be purified using a combination of chromatography on astrong cation exchanger followed by affinity chromatography andsize-exclusion chromatography.

As shown in more detail in the examples that follow, zvegf4 is highlyexpressed in the kidney, and over-expression of zvegf4 in mice byinjection of an adenovirus vector encoding zvegf4 elicitsfibroproliferative changes in the kidney. Two readily identifiablefeatures of these changes are (a) enlarged glomeruli due in part tomesangial cell proliferation, and (b) tubular regeneration due to tubuleepithelial cell proliferation. These findings indicate that an increasein zvegf4 protein can modify the function of, and the interactionsamong, mesangial, epithelial, endothelial, smooth muscle, andinterstitial cells, which are all key players in glomerular and vasculardiseases of the kidney. Furthermore, zvegf4 has been found to affectcell proliferation in at least some of these cells in vitro. Experimentshave also shown that the activity of zvegf4 is mediated by two PDGFreceptor subunits, alpha and beta (PDGF-αR and PDGF-βR). These receptorsubunits are widely expressed in most renal cell types, and theirexpression is upregulated in a number of kidney pathologies (e.g., Iidaet al., Proc. Natl. Acad. Sci. USA 88:6560–6564, 1991). Stimulation ofPDGF receptors has been implicated in fibroproliferative diseases of thekidney in a variety of animal models (e.g., Ooi et al., P.S.E.B.M.213:230–237, 1996; Lindahl et al., Development 125:3313–3322, 1998;Lindahl and Betsholtz, Curr. Op. Nephr. Hypert. 7:21–26, 1998; andBetsholtz and Raines, Kidney Int. 51:1361–1369, 1997).

As also shown herein, zvegf4 has been found to stimulate the productionof TGF-β by rat liver stellate cells. TGF-β is thought to be a majormediator of fibrosis, due to its ability to stimulate extracellularmatrix synthesis (especially collagen and fibronectin) in a variety ofmesenchymal cell types, most notably fibroblasts. TGF-β has beenimplicated in the development of fibrosis of the heart, lung, liver, andkidney (Ledbetter et al., Kidney Int. 58(6):2367–2376, 2000; Chen etal., Mol. Cell Cardiol. 32(10):1805–1819, 2000; Nakamura et al.,Hepatology 32(2):247–255, 2000; Martin et al., Int. J. Radiat. Oncol.Biol. Phys. 47(2):277–290, 2000; Sanderson et al., Proc. Natl. Acad.Sci. USA 92(7):2572–2576, 1995). Increased expression of zvegf4 inorgans such as the heart, kidney, lung or liver may result in fibrosis,which may at least in part be mediated and exacerbated by the enhancedproduction of TGF-β.

Zvegf4 has been found to be highly expressed in mouse prostate tumorcell lines as shown by Northern blotting. In addition, animals treatedwith a zvegf4-encoding adenovirus vector displayed invasion of themarrow space by endosteal bone, indicating an effect of zvegf4 on bonegrowth. In view of the high incidence of bony metastases in mensuffering from prostate cancer, these results implicate zvegf4 as amediator of prostate tumor-related cancellous bone growth.

Additional evidence indicates that zvegf4 may bind to cell-surfacesemaphorins, presumably via the CUB domain. Cells having cell-surfacesemaphorins include endothelial cells, neuronal cells, lymphocytes, andvarious tumor cells.

In view of the experiments summarized above and disclosed in more detailherein, it is expected that altered zvegf4 expression may initiate orexacerbate renal disease and other fibroproliferative disorders. In thiscontext, inhibiting the action of zvegf4 using a zvegf4 antagonist willlimit the progress of such disorders. Zvegf4 antagonists include,without limitation, anti-zvegf4 antibodies (including neutralizingantibodies), soluble zvegf4 receptors (including soluble PDGF betareceptor; see, e.g., Herren et al., J. Biol. Chem. 268:15088–15095,1993; U.S. Pat. No. 6,018,026; and soluble PDGF alpha receptor),anti-receptor antibodies, and other peptidic and non-peptidic agents,including ribozymes, antisense polynucleotides, small moleculeinhibitors, and mitogenically inactive, receptor-binding zvegf4polypeptides.

Within the present invention zvegf4 antagonists are used to block theproliferative or profibrotic effects of zvegf4. Thus, the presentinvention provides methods of inhibiting, reducing, preventing, orotherwise treating fibroproliferative disorders, including, withoutlimitation, scar formation, keloids, scleroderma, liver fibrosis, lungfibrosis, kidney fibrosis, myelofibrosis, post-surgical fibroticadhesions, fibrotic tumors, fibroproliferative disorders of thevasculature, fibroproliferative disorders of the prostate,fibroproliferative disorders of bone, fibromatosis, fibroma,fibrosarcoma, and the like.

Fibroproliferative disorders of the kidney include, without limitation,glomerulonephritis (including membranoproliferative, diffuseproliferative, rapidly progressive, and chronic forms), diabeticglomerulosclerosis, focal glomerulosclerosis, diabetic nephropathy,lupus nephritis, tubulointerstitial fibrosis, membranous nephropathy,amyloidosis (which affects the kidney among other tissues), renalarteriosclerosis, and nephrotic syndrome. The glomerulus is a majortarget of many types of renal injury, including immunologic (e.g.,immune-complex- or T-cell-mediated), hemodynamic (systemic or renalhypertension), metabolic (e.g., diabetes), “atherosclerotic”(accumulation of lipids in the glomerulus), infiltrative (e.g.,amyloid), and toxic (e.g., snake venom) injuries (Johnson, Kidney Int.45:1769–1782, 1994). The renal structural changes in patients withdiabetic nephropathy include hypertrophy of the glomerulus, thickeningof the glomerular and tubular membranes (due to accumulated matrix), andincreased amounts of matrix in the measangium and tubulointerstitium(Ziyadeh et al., Proc. Natl. Acad. Sci. USA 97:8015–8020, 2000).Glomerular hypertension due to intrarenal hemodynamic changes indiabetes can contribute to the progression of diabetic nephropathy(Ishida et al., Diabetes 48:595–602, 1999). Autoimmune nephritis canalso lead to altered mesangial cell growth responses (Liu and Ooi, J.Immunol. 151:2247–2251, 1993). Infection by hepatitis-C virus can alsoresult in idiopathic membranoproliferative glomerulonephritis (Johnsonet al., N. Engl. J. Med. 328:465–470, 1993).

Fibroproliferative disorders of the lung include, for example,silicosis, asbestosis, idiopathic pulmonary fibrosis, bronchiolitisobliterans-organizing pneumonia, pulmonary fibrosis associated withhigh-dose chemotherapy, idiopathic pulmonary fibrosis, and pulmonaryhypertension. These diseases are characterized by cell proliferation andincreased production of extracellular matrix components, such ascollagens, elastin, fibronectin, and tenascin-C.

Fibrosis of the liver can result from damage due to chronic liverdisease, including chronic active hepatitis (including hepatitis C) andmany other types of cirrhosis. Widespread, massive necrosis, includingdestruction of virtually the entire liver, can be caused by, inter alia,fulminant viral hepatitis; overdoses of the analgesic acetaminophen;exposure to other drugs and chemicals such as halothane, monoamineoxidase inhibitors, agents employed in the treatment of tuberculosis,phosphorus, carbon tetrachloride, and other industrial chemicals.Conditions associated with ultrastructural lesions that do notnecessarily produce obvious liver cell necrosis include Reye's syndromein children, tetracycline toxicity, and acute fatty liver of pregnancy.Cirrhosis, a diffuse process characterized by fibrosis and a conversionof normal architecture into structurally abnormal nodules, can comeabout for a variety reasons including alcohol abuse, post necroticcirrhosis (usually due to chronic active hepatitis), biliary cirrhosis,pigment cirrhosis, cryptogenic cirrhosis, Wilson's disease, andalpha-1-antitrypsin deficiency. In cases of liver fibrosis it may bebeneficial to administer a zvegf4 antagonist to suppress the activationof stellate cells, which have been implicated in the production ofextracellular matrix in fibrotic liver (Li and Friedman, J.Gastroenterol. Hepatol. 14:618–633, 1999).

Diseases of the skeleton that are due to modified growth and matrixproduction in the bone include, but are not limited to, osteopetrosis,hyperostosis, osteosclerosis, osteoarthritis, and ectopic bone formationin metastatic prostate cancer. Fibroproliferative disorders of bone arecharacterized by aberrant and ectopic bone formation, commonly seen asactive proliferation of the major cell types participating in boneformation as well as elaboration by those cells of a complex bonematrix. Exemplary of such bone disorders is the fibrosis that occurswith prostate tumor metastases to the axial skeleton. In prostatetumor-related cancellous bone growth, prostate carcinoma cells caninteract reciprocally with osteoblasts to produce enhanced tumor growthand osteoblastic action when they are deposited in bone (Zhau et al.,Cancer 88:2995–3001, 2000; Ritchie et al., Endocrinology 138:1145–1150,1997). As disclosed in more detail below, mice receiving azvegf4-encoding adenovirus vector displayed a similar pathology as thatobserved in prostate cancer patients who display tumor metastases in theaxial skeleton and consequent formation of endosteal bone. In addition,a panel of mouse prostate cell lines (epithelial and stromal) propagatedin culture were found to express very high levels of zvegf4 messengerRNA. These data suggest that zvegf4 is involved (via autocrine and/orparacrine mechanisms) in prostate tumor growth, metastasis, and effectsin bone. Fibroproliferative responses of the bone originating in theskeleton per se include osteopetrosis and hyperstosis. A defect inosteoblast differentiation and function is thought to be a major causein osteopetrosis, an inherited disorder characterized by bone sclerosisdue to reduced bone resorption, wherein marrow cavities fail to develop,resulting in extramedullary hematopoiesis and severe hematologicabnormalities associated with optic atrophy, deafness, and mentalretardation (Lajeunesse et al., J. Clin Invest. 98:1835–1842, 1996). Inosteoarthritis, bone changes are known to occur, and bone collagenmetabolism is increased within osteoarthritic femoral heads. Thegreatest changes occur within the subchondral zone, supporting a greaterproportion of osteoid in the diseased tissue (Mansell and Bailey, J.Clin. Invest. 101:1596–1603, 1998).

Fibroproliferative disorders of the vasculature include, for example,transplant vasculopathy, which is a major cause of chronic rejection ofheart transplantation. Transplant vasculopathy is characterized byaccelerated atherosclerotic plaque formation with diffuse occlusion ofthe coronary arteries, which is a “classic” fibroproliferative disease.See, Miller et al., Circulation 101:1598–1605, 2000).

Antibodies used as zvegf4 antagonists include antibodies thatspecifically bind to a zvegf4 protein or a zvegf4 cell-surface receptorand, by so binding, reduce or prevent the binding of zvegf4 protein tothe receptor and, consequently, reduce or block the receptor-mediatedactivity of zvegf4. As used herein, the term “antibodies” includespolyclonal antibodies, affinity-purified polyclonal antibodies,monoclonal antibodies, and antigen-binding fragments, such as F(ab′)₂and Fab proteolytic fragments. Genetically engineered intact antibodiesor fragments, such as chimeric antibodies, Fv fragments, single chainantibodies, and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Monoclonal antibodies can also beproduced in mice that have been genetically altered to produceantibodies that have a human structure.

Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Cooligan et al. (eds.),Current Protocols in Immunology, National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor, N.Y., 1989; andHurrell (ed.), Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press. Inc., Boca Raton, Fla., 1982. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated by inoculating a variety of warm-blooded animals such ashorses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats witha zvegf4 polypeptide or a fragment thereof.

Immunogenic polypeptides will comprise an epitope-bearing portion of azvegf4 polypeptide (e.g., as shown in SEQ ID NO:2) or receptor. An“epitope” is a region of a protein to which an antibody can bind. See,for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002,1984. Epitopes can be linear or conformational, the latter beingcomposed of discontinuous regions of the protein that form an epitopeupon folding of the protein. Linear epitopes are generally at least 6amino acid residues in length. Relatively short synthetic peptides thatmimic part of a protein sequence are routinely capable of eliciting anantiserum that reacts with the partially mimicked protein. See,Sutcliffe et al., Science 219:660–666, 1983. Immunogenic,epitope-bearing polypeptides contain a sequence of at least six, oftenat least nine, more often from 15 to about 30 contiguous amino acidresidues of a zvegf4 protein or receptor. Polypeptides comprising alarger portion of a zvegf4 protein or receptor, i.e. from 30 to 50residues up to the entire sequence are included. It is preferred thatthe amino acid sequence of the epitope-bearing polypeptide is selectedto provide substantial solubility in aqueous solvents, that is thesequence includes relatively hydrophilic residues, and hydrophobicresidues are substantially avoided (see FIGS. 1A–1G). Such regions ofSEQ ID NO:2 include, for example, residues 39–44, 252–257, 102–107,264–269, and 339–344. Exemplary longer peptide immunogens includepeptides comprising residues (i) 131–148, (ii) 230–253, or (iii) 333–355of SEQ ID NO:2. Peptide (ii) can be prepared with an additionalC-terminal Cys residue and peptide (iii) with an additional N-terminalCys residue to facilitate coupling.

The immunogenicity of a polypeptide immunogen may be increased throughthe use of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of a zvegf4polypeptide or a portion thereof with an immunoglobulin polypeptide orwith maltose binding protein. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA), or tetanus toxoid) for immunization.

Alternative techniques for generating or selecting antibodies include invitro exposure of lymphocytes to a polypeptide immunogen, and selectionof antibody display libraries in phage or similar vectors (for instance,through use of immobilized or labeled polypeptide). Techniques forcreating and screening such random peptide display libraries are knownin the art (e.g., Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al.,U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 andLadner et al., U.S. Pat. No. 5,571,698), and random peptide displaylibraries and kits for screening such libraries are availablecommercially, for instance from Clontech Laboratories (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zvegf4sequences disclosed herein to identify proteins that bind to zvegf4.

Antibodies are determined to be specifically binding if they bind totheir intended target (e.g., zvegf4 protein or receptor) with anaffinity at least 10-fold greater than the binding affinity to control(e.g., non-zvegf4 or non-receptor) polypeptide or protein. In thisregard, a “non-zvegf4 polypeptide” includes the related molecules VEGF,VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A, and PDGF-B, but excludes zvegf4polypeptides from non-human species. Due to the high level of amino acidsequence identity expected between zvegf4 orthologs, antibodies specificfor human zvegf4 may also bind to zvegf4 from other species. The bindingaffinity of an antibody can be readily determined by one of ordinaryskill in the art, for example, by Scatchard analysis (Scatchard, G.,Ann. NY Acad. Sci. 51: 660–672, 1949). Methods for screening andisolating specific antibodies are well known in the art. See, forexample, Paul (ed.), Fundamental Immunology, Raven Press, 1993; Getzoffet al., Adv. in Immunol. 43:1–98, 1988; Goding (ed.), MonoclonalAntibodies: Principles and Practice, Academic Press Ltd., 1996; andBenjamin et al., Ann. Rev. Immunol. 2:67–101, 1984.

A variety of assays known to those skilled in the art can be utilized todetect antibodies that specifically bind to zvegf4 proteins orreceptors. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassays, inhibition or competition assays, and sandwich assays.

For therapeutic applications it is generally preferred to useneutralizing antibodies. As used herein, the term “neutralizingantibody” denotes an antibody that inhibits at least 50% of thebiological activity of the cognate antigen when the antibody is added ata 1000-fold molar access. Those of skill in the art will recognize thatgreater neutralizing activity is sometimes desirable, and antibodiesthat provide 50% inhibition at a 100-fold or 10-fold molar access may beadvantageously employed.

Zvegf4 antagonists also include soluble receptors. As used herein, a“soluble zvegf4 receptor” is a ligand-binding zvegf4 receptorpolypeptide that is not bound to a cell membrane. Soluble receptors aremost commonly receptor polypeptides that comprise at least a portion ofthe extracellular, ligand binding domain sufficient to bind ligand butlack transmembrane and cytoplasmic domains. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Receptor polypeptides are said to be substantially free oftransmembrane and intracellular polypeptide segments when they lacksufficient portions of these segments to provide membrane anchoring orsignal transduction, respectively. Soluble receptors can compriseadditional amino acid residues, such as affinity tags that provide forpurification of the polypeptide or provide sites for attachment of thepolypeptide to a substrate, or immunoglobulin constant region sequences.Dimeric and higher order multimeric soluble receptors are preferred fortheir ability to bind ligand with high affinity. A soluble receptor canbe prepared as a fusion to a dimerizing protein as disclosed in U.S.Pat. Nos. 5,155,027 and 5,567,584. Dimerizing proteins in this regardinclude, for example, immunoglobulin fragments comprising constantregion and hinge domains (e.g., IgG Fc fragments).

Zvegf4 antagonists further include antisense polynucleotides, which canbe used to inhibit zvegf4 gene transcription and thereby inhibit cellactivation and/or proliferation in vivo. Polynucleotides that arecomplementary to a segment of a zvegf4-encoding polynucleotide (e.g., apolynucleotide as set forth in SEQ ID NO:1) are designed to bind tozvegf4-encoding mRNA and to inhibit translation of such mRNA. Antisensepolynucleotides can be targetted to specific tissues using a genetherapy approach with specific vectors and/or promoters, such as viraldelivery systems as disclosed in more detail below.

Ribozymes can also be used as zvegf4 antagonists within the presentinvention. Ribozymes are RNA molecules that contains a catalytic centerand a target RNA binding portion. The term includes RNA enzymes,self-splicing RNAs, self-leaving RNAs, and nucleic acid molecules thatperform these catalytic functions. A ribozyme selectively binds to atarget RNA molecule through complementary base pairing, bringing thecatalytic center into close proximity with the target sequence. Theribozyme then cleaves the target RNA and is released, after which it isable to bind and cleave additional molecules. A nucleic acid moleculethat encodes a ribozyme is termed a “ribozyme gene.” Ribozymes can bedesigned to express endonuclease activity that is directed to a certaintarget sequence in a mRNA molecule (see, for example, Draper andMacejak, U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson andGoldberg, U.S. Pat. No. 5,225,337). An expression vector can beconstructed in which a regulatory element is operably linked to anucleotide sequence that encodes a ribozyme.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode azvegf4 polypeptide. According to this approach, an external guidesequence can be constructed for directing the endogenous ribozyme, RNaseP, to a particular species of intracellular mRNA, which is subsequentlycleaved by the cellular ribozyme (see, for example, Altman et al., U.S.Pat. No. 5,168,053; Yuan et al., Science 263:1269, 1994; Pace et al.,WIPO Publication No. WO 96/18733; George et al., WIPO Publication No. WO96/21731; and Werner et al., WIPO Publication No. WO 97/33991). Anexternal guide sequence generally comprises a ten- to fifteen-nucleotidesequence complementary to zvegf4 mRNA, and a 3′-NCCA nucleotidesequence, wherein N is preferably a purine. The external guide sequencetranscripts bind to the targeted mRNA species by the formation of basepairs between the mRNA and the complementary external guide sequences,thus promoting cleavage of mRNA by RNase P at the nucleotide located atthe 5′-side of the base-paired region.

The growth factor domain of zvegf4 has been found to be an activespecies of the molecule that binds to cell-surface PDGF receptorscompetitively with other PDGF isoforms. Proteolytic processing to removethe N-terminal portion of the molecule is required for this activity.Thus, inhibitors of this proteolytic activation can also be used aszvegf4 antagonists within the present invention.

For pharmaceutical use, zvegf4 antagonists are formulated for topical orparenteral, particularly intravenous or subcutaneous, delivery accordingto conventional methods. In general, pharmaceutical formulations willinclude a zvegf4 antagonist in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. A “therapeutically effective amount” of acomposition is that amount that produces a statistically significanteffect, such as a statistically significant reduction in diseaseprogression or a statistically significant improvement in organfunction. The exact dose will be determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The therapeuticformulations will generally be administered over the period required toachieve a beneficial effect, commonly up to several months and, intreatment of chronic conditions, for a year or more. Dosing is daily orintermittently over the period of treatment. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. Sustained release formulations can also be employed. Fortreatment of pulmonary fibrosis, a zvegf4 antagonist can be delivered byaerosolization according to methods known in the art. See, for example,Wang et al., U.S. Pat. No. 5,011,678; Gonda et al., U.S. Pat. No.5,743,250; and Lloyd et al., U.S. Pat. No. 5,960,792.

Other mitogenic factors, including the PDGFs, EGF, TGF-β1 and TGF-β2,and FGFs, have been implicated in the initiation or perpetuation offibrosis. It may therefore be advantageous to combine a zvegf4antagonist with one or more antagonists of these other factors.

Antibodies are preferably administered parenterally, such as by bolusinjection or infusion (intravenous, intramuscular, intraperitoneal, orsubcutaneous) over the course of treatment. Antibodies are generallyadministered in an amount sufficient to provide a minimum circulatinglevel of antibody throughout the treatment period of betweenapproximately 20 μg and 1 mg/kg body weight. In this regard, it ispreferred to use antibodies having a circulating half-life of at least12 hours, preferably at least 4 days, more preferably up to 14–21 days.Chimeric and humanized antibodies are expected to have circulatoryhalf-lives of up to four and up to 14–21 days, respectively. In manycases it will be preferable to administer daily doses during a hospitalstay, followed by less frequent bolus injections during a period ofoutpatient treatment. Antibodies can also be delivered by slow-releasedelivery systems, pumps, and other known delivery systems for continuousinfusion. Dosing regimens may be varied to provide the desiredcirculating levels of a particular antibody based on itspharmacokinetics. Thus, doses will be calculated so that the desiredcirculating level of therapeutic agent is maintained. Daily dosesreferred to above may be administered as larger, less frequent bolusadministrations to provide the recited dose averaged over the term ofadministration.

Those skilled in the art will recognize that the same principles willguide the use of other zvegf4 antagonists. The dosing regimen for agiven antagonist will be determined by a number of factors includingpotency, pharmacokinetics, and the physicochemical nature of theantagonist. For example, non-peptidic zvegf4 antagonists may beadministered enterally.

Therapeutic polynucleotides, such as antisense polynucleotides, can bedelivered to patients or test animals by way of viral delivery systems.Exemplary viruses for this purpose include adenovirus, herpesvirus,retroviruses, vaccinia virus, and adeno-associated virus (AAV).Adenovirus, a double-stranded DNA virus, is currently the best studiedgene transfer vector for delivery of heterologous nucleic acids. Forreview, see Becker et al., Meth. Cell Biol. 43:161–189, 1994; andDouglas and Curiel, Science & Medicine 4:44–53, 1997. The adenovirussystem offers several advantages. Adenovirus can (i) accommodaterelatively large DNA inserts; (ii) be grown to high-titer; (iii) infecta broad range of mammalian cell types; and (iv) be used with manydifferent promoters, including ubiquitous, tissue specific, andregulatable promoters. Because adenoviruses are stable in thebloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. When intravenouslyadministered to intact animals, adenovirus primarily targets the liver.If the adenoviral delivery system has an E1 gene deletion, the viruscannot replicate in the host cells. However, the host's tissue (e.g.,liver) will express and process (and, if a signal sequence is present,secrete) the heterologous protein.

An alternative method of gene delivery comprises removing cells from thebody and introducing a vector into the cells as a naked DNA plasmid. Thetransformed cells are then re-implanted in the body. Naked DNA vectorsare introduced into host cells by methods known in the art, includingtransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem.263:14621–14624, 1988; Wu et al., J. Biol. Chem. 267:963–967, 1992; andJohnston and Tang, Meth. Cell Biol. 43:353–365, 1994.

Zvegf4 antagonists can be analyzed for receptor-binding activity orinhibition of zvegf4-receptor binding by a variety of methods that arewell known in the art, including receptor competition assays (Bowen-Popeand Ross, Methods Enzymol, 109:69–100, 1985) and through the use ofsoluble receptors, including receptors produced as IgG fusion proteins(U.S. Pat. No. 5,750,375). Receptor-binding assays can be performed oncell lines that contain cell-surface receptors for zvegf4. The receptorscan be naturally present in the cell, or can be recombinant receptorsexpressed by genetically engineered cells.

Activity of zvegf4 antagonists can be measured in vitro using culturedcells in assays designed to measure zvegf4 activity. Antagonists willreduce the effects of zvegf4 within the assay. Mitogenic activity can bemeasured using known assays, including ³H-thymidine incorporation assays(as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749–773,1985 and Wahl et al., Mol. Cell Biol. 8:5016–5025, 1988), dyeincorporation assays (as disclosed by, for example, Mosman, J. Immunol.Meth. 65:55–63, 1983 and Raz et al., Acta Trop. 68:139–147, 1997) orcell counts. Exemplary mitogenesis assays measure incorporation of³H-thymidine into (1) 20% confluent cultures to look for the ability ofzvegf4 proteins to further stimulate proliferating cells, and (2)quiescent cells held at confluence for 48 hours to look for the abilityof zvegf4 proteins to overcome contact-induced growth inhibition.Exemplary dye incorporation assays include measurement of theincorporation of the dye Alamar blue (Raz et al., ibid.) into targetcells. See also, Gospodarowicz et al., J. Cell. Biol. 70:395–405, 1976;Ewton and Florini, Endocrinol. 106:577–583, 1980; and Gospodarowicz etal., Proc. Natl. Acad. Sci. USA 86:7311–7315, 1989.

The biological activities of zvegf4 antagonists can be studied innon-human animals by administration of exogenous compounds, byexpression of zvegf4 inhibitory polynucleotides, and by suppression ofendogenous zvegf4 expression through knock-out techniques. Viraldelivery systems (disclosed above) can be employed. Zvegf4 antagonistscan be administered or expressed individually, in combination with otherzvegf4 antagonists, or in combination other compounds, including othergrowth factor antagonists. Test animals are monitored for changes insuch parameters as clinical signs, body weight, blood cell counts,clinical chemistry, histopathology, and the like.

Effects of zvegf4 antagonists on liver and kidney fibrosis can be testedin known animal models, such as the db/db mouse model disclosed by Cohenet al., Diabetologia 39:270–274, 1996 and Cohen et al., J. Clin. Invest.95:2338–2345, 1995, or transgenic animal models (Imai et al., Contrib.Nephrol. 107:205–215, 1994).

Effects on lung fibrosis can also be assayed in a mouse model usingbleomycin. The chemotherapy agent bleomycin is a known causative agentof pulmonary fibrosis in humans and can induce interstitial lung diseasein mice, including an increase in the number of fibroblasts, enhancedcollagen deposition, and dysregulated matrix remodeling. C57B1/6 miceare administered bleomycin by osmotic minipump for 1 week. There followsa period of inflammation, with cutaneous toxicity beginningapproximately 4–7 days after bleomycin administration and continuing forabout a week, after which the mice appear to regain health. About 3–4weeks after the finish of bleomycin delivery, the mice are sacrificed,and the lungs are examined histologically for signs of fibrosis. Scoringis based on the extent of lung fibrotic lesions and their severity.Serum is assayed for lactic dehydrogenase, an intracellular enzyme thatis released into the circulation upon general cell death or injury. Lungtissue is assayed for hydroxyproline as a measure of collagendeposition.

Mice and other animals carrying a zvegf4-expressing adenovirus vectorare also useful models for testing zvegf4 antagonists and otherantifibroproliferative agents.

The invention is further illustrated by the following, non-limitingexamples.

EXAMPLES Example 1

Zvegf4 was identified from the sequence of a clone from a human chronicmyelogenous leukemia cell (K562) library by its homology to the VEGFfamily. Additional sequence was elucidated from a long sequence read ofa clone from a pituitary library. An antisense expressed sequence tag(EST) for zvegf4 was found, for which its 5′ partner was identified.This 5′ EST (EST448186; GenBank) appeared to contain the 5′ untranslatedsequence for zvegf4. A primer was designed from EST448186 to close thegap in the sequence. 20 pm each of oligonucleotides ZC21,987 (SEQ IDNO:5) and ZC21,120 (SEQ ID NO:6), and 1.93 μg of a thyroid library wereused in the PCR reaction with 5% DMSO and 1/10 volume of a commercialreagent (GC-MELT; Clontech Laboratories, Inc., Palo Alto, Calif.). Thereaction was run for 1 minute at 94 degrees; then 30 cycles of 94degrees, 20 seconds; 67 degrees, 1 minute; then a final 5-minuteincubation at 72 degrees. A resulting 833-bp product was sequenced andfound to be a zvegf4 fragment containing the remainder of the codingsequence with an intiation MET codon, upstream stop codon, and 5′untranslated sequence. The composite sequence included an open readingframe of 1,110 bp (SEQ ID NO:1).

Example 2

A partial mouse zvegf4 sequence was obtained by probing a mouse genomiclibrary (obtained from Clontech Laboratories, Inc.) with a 1,289 bpEcoRI human zvegf4 restriction digest fragment containing the entirecoding sequence. The probe was labeled with ³²P using a commerciallyavailable kit (REDIPRIME II random-prime labeling system; AmershamPharmacia, Buckinghamshire, England). Unincorporated radioactivity wasremoved using a commercially available push column (NUCTRAP column;Stratagene, La Jolla, Calif.; see U.S. Pat. No. 5,336,412). Twenty-fourfilter lifts were prehybridized overnight at 50° C. in a hybridizationsolution (EXPRESSHYB Hybridization Solution; Clontech Laboratories,Inc.) containing 0.1 mg/ml salmon sperm DNA that had been boiled 5minutes, then iced. Filters were hybridized overnight at 50° C. inhybridization solution (EXPRESSHYB) containing 1.0×10⁶ cpm/ml zvegf4probe, 0.1 mg/ml salmon sperm DNA, and 0.5 μg/ml mouse cot-1 DNA thathad been boiled 5 minutes, then iced. Filter lifts were washed in 2×SSC,0.1% SDS at room temperature for 2 hours, then the temperature wasraised to 60° C. for one hour. Overnight exposure at −80° C. showed 7putative primary hits.

Four of the primary hits were plated on a lawn of E. coli K802 cells(obtained from Clontech Laboratories, Inc.). Filter lifts were preparedand hybridized overnight with the human zvegf4 probe. Two of the 4primary putative hits that were tested came up positive.

DNA was prepared from one positive plaque and digested with BamHI andPstI. The digest was run on a 1% Tris-Borate-EDTA gel, and a 2.0 kbdoublet and 2.7 kb/2.9 kb bands were excised from the gel and extractedfrom the agarose by conventional methods. Both 2.0 kb fragments werefound to strongly hybridize to the human zvegf4 probe. These fragmentswere sequenced and found to contain part of the mouse zvegf4 CUB domain.Primers were designed from the sequence for use in a PCR cDNA screen.

A panel of mouse cDNAs was screened by PCR with primers ZC26,317 (SEQ IDNO:7) and ZC26,318 (SEQ ID NO:8). Embryo, salivary gland, neonatal skin,and testis showed strong products of the predicted 200 bp size.

Mouse testis and salivary gland libraries were screened by PCR usingprimers ZC26,317 (SEQ ID NO:7) and ZC26,318 (SEQ ID NO:8). The testislibrary yielded one clone, named “zvegf4mpzp7x-6”, that was incompleteat the 5′ end and appeared to contain an intron at the 5′ end. Thesalivary gland library yielded one clone, named “zvegf4mpzp7x-7”, thathad a 225-bp deletion in coding compared to clone zvegf4mpzp7x-6. Thesequences derived from zvegf4mpzp7x-6 and zvegf4mpzp7x-7 were combinedto produce a full-length mouse zvegf4 polynucleotide sequence (SEQ IDNO:3) and mouse zvegf4 polypeptide sequence (SEQ ID NO:4).

A full-length cDNA clone was generated by a two-step ligation offragments from the two clones. An EcoRI/HindIII 3′ fragment was preparedfrom clone zvegf4mpzp7x-6. The 528-bp fragment was gel-purified andligated into a phagemid vector (PBLUESCRIPT II KS(+); Stratagene) thathad been digested with EcoRI and HindIII. Three μg of the resultingconstruct was digested with 15 units of EcoRI. The linearized plasmidwas purified and ligated with a 754-bp 5′ EcoRI fragment from clonezvegf4mpzp7x-7.

Example 3

Recombinant human zvegf4 having a carboxyl-terminal Glu-Glu affinity tagwas produced in a baculovirus expression system according toconventional methods. The culture was harvested, and the cells werelysed with a solution of 0.02 M Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM DTT, 1mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (PEFABLOC SC;Boehringer-Mannheim), 0.5 μM aprotinin, 4 mM leupeptin, 4 mM E-64, 1%NP-40 at 4° C. for 15 minutes on a rotator. The solution wascentrifuged, and the supernatant was recovered. Twenty ml of extract wascombined with 50 μl of anti-Glu-Glu antibody conjugated to derivatizedagarose beads (SEPHAROSE; Amersham Pharmacia Biotech Inc., Piscataway,N.J.) in 50 μl buffer. The mixture was incubated on a rotator at 4° C.overnight. The beads were recovered by centrifugation and washed 3×15minutes at 4° C. Pellets were combined with sample buffer containingreducing agent and heated at 98° C. for five minutes. The protein wasanalyzed by polyacrylamide gel electrophoresis under reducing conditionsfollowed by western blotting on a PVDF membrane using an antibody to theaffinity tag. Two bands were detected, one a M_(r)≈49 kD and the otherat M_(r)≈21 kD. Sequence analysis showed the larger band to comprise twosequences, one beginning at Arg-19 of SEQ ID NO:2 and the otherbeginning at Asn-35 of SEQ ID NO:2. The asparagine residue appeared tohave been deamidated to an aspartic acid. The smaller band began atSer-250 of SEQ ID NO:2.

Example 4

To prepare adenovirus vectors, the protein coding region of zvegf4 isamplified by PCR using primers that add FseI and AscI restriction sitesat the 5′ and 3′ termini, respectively. PCR primers are used with atemplate containing the full-length zvegf4 cDNA in a PCR reaction asfollows: incubation at 95° C. for 5 minutes; followed by 15 cycles at95° C. for 1 min., 58° C. for 1 min., and 72° C. for 1.5 min.; followedby 72° C. for 7 min.; followed by a 4° C. soak. The reaction productsare loaded onto a 1.2% low-melt agarose (SEAPLAQUE GTG™; FMC, Rockland,Me.) gel in TAE buffer. The zvegf4 PCR product is excised from the geland purified using a spin column containing a silica gel membrane(QIAQUICK Gel Extraction Kit; Qiagen, Inc., Valencia, Calif.) as per kitinstructions. The zvegf4 product is then digested, phenol/chloroformextracted, EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA pH8). The zvegf4 fragment is then ligated into the cloning sites of thetransgenic vector pTG12-8. Vector pTG12-8 was derived from p2999B4(Palmiter et al., Mol. Cell Biol. 13:5266–5275, 1993) by insertion of arat insulin II intron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I)into the Nru I site. The vector comprises a mouse metallothionein (MT-1)promoter (ca. 750 bp) and human growth hormone (hGH) untranslated regionand polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5′flanking sequence and 7 kb of MT-1 3′ flanking sequence. The constructis transformed into E. coli host cells (ELECTROMAX DH10B™ cells;obtained from Life Technologies, Inc., Gaithersburg, Md.) byelectroporation. Clones containing zvegf4 DNA are identified byrestriction analysis. A positive clone is confirmed by directsequencing.

The zvegf4 cDNA is released from the pTG12-8 vector using FseI and AscIenzymes. The cDNA is isolated on a 1% low melt agarose gel, and is thenexcised from the gel. The gel slice is melted at 70° C., extracted twicewith an equal volume of Tris-buffered phenol, and EtOH precipitated. TheDNA is resuspended in 10 μl H₂O.

The zvegf4 cDNA is cloned into the FseI-AscI sites of a modifiedpAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509–2514, 1998).This construct contains a green fluorescent protein (GFP) marker gene.The CMV promoter driving GFP expression has been replaced with the SV40promoter, and the SV40 polyadenylation signal has been replaced with thehuman growth hormone polyadenylation signal. In addition, the nativepolylinker has been replaced with FseI, EcoRV, and AscI sites. Thismodified form of pAdTrack CMV is named pZyTrack. Ligation is performedusing a DNA ligation and screening kit (FAST-LINK; EpicentreTechnologies, Madison, Wis.). In order to linearize the plasmid,approximately 5 μg of the pZyTrack zvegf4 plasmid is digested with PmeI.Approximately 1 μg of the linearized plasmid is cotransformed with 200ng of super coiled pAdEasy (He et al., ibid.) into BJ5183 cells. Theco-transformation is done using a Bio-Rad Gene Pulser at 2.5 kV, 200ohms and 25 μF. The entire co-transformation is plated on 4 LB platescontaining 25 μg/ml kanamycin. The smallest colonies are picked andexpanded in LB/kanamycin, and recombinant adenovirus DNA is identifiedby standard DNA miniprep procedures. Digestion of the recombinantadenovirus DNA with FseI and AscI confirms the presence of zvegf4 DNA.The recombinant adenovirus miniprep DNA is transformed into E. coliDH10B competent cells, and DNA is prepared therefrom.

Approximately 5 μg of recombinant adenoviral DNA is digested with PacIenzyme (New-England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20–30U of PacI. The digested DNA is extracted twicewith an equal volume of phenol/chloroform and precipitated with ethanol.The DNA pellet is resuspended in 10 μl distilled water. A T25 flask ofQBI-293A cells (Quantum Biotechnologies, Inc., Montreal, Canada),inoculated the day before and grown to 60–70% confluence, aretransfected with the PacI digested DNA. The PacI-digested DNA is dilutedup to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mMHEPES). In a separate tube, 20 μl 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate(DOTAP; Boehringer Mannheim) is diluted to a total volume of 100 μl withHBS. The DNA is added to the DOTAP, mixed gently by pipeting up anddown, and left at room temperature for 15 minutes. The media is removedfrom the 293A cells and washed with 5 ml serum-free MEM-alpha (LifeTechnologies, Gaithersburg, Md.) containing 1 mM sodium pyruvate (LifeTechnologies), 0.1 mM MEM non-essential amino acids (Life Technologies)and 25 mM HEPES buffer (Life Technologies). 5 ml of serum-free MEM isadded, and the cells are held at 37° C. The DNA/lipid mixture is addeddrop-wise to the flask, mixed gently, and incubated at 37° C. for 4hours. After 4 hours the media containing the DNA/lipid mixture isaspirated off and replaced with 5 ml complete MEM containing 5% fetalbovine serum. The transfected cells are monitored for GFP expression andformation of foci (viral plaques).

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, cells expressing GFP start to form foci. The crude virallysate is harvested by using a cell scraper to collect the cells. Thelysate is transferred to a 50 ml conical tube. To release most of thevirus particles from the cells, three freeze/thaw cycles are done in adry ice/ethanol bath and a 37° C. waterbath.

Ten 10-cm plates of nearly confluent (80–90%) 293A cellsare set up 20hours prior to infection. The crude lysate is amplified (primaryamplification) to obtain a working stock of zvegf4 recombinantadenovirus (rAdV) lysate. 200 ml of crude rAdV lysate is added to each10-cm plate, and the plates are monitored for 48 to 72 hours looking forcytopathic effect (CPE) under the white light microscope and expressionof GFP under the fluorescent microscope. When all of the cells show CPE,this 1° stock lysate is collected, and freeze/thaw cycles are performedas described above.

Secondary (2°) amplification of zvegf4 rAdV is obtained from twenty15-cm tissue culture dishes of 80–90% confluent 293A cells. All but 20ml of 5% MEM media is removed, and each dish is inoculated with 300–500ml of 1° amplified rAdv lysate. After 48 hours the cells are lysed fromvirus production, the lysate is collected into 250 ml polypropylenecentrifuge bottles, and the rAdV is purified.

NP-40 detergent is added to a final concentration of 0.5% to the bottlesof crude lysate to lyse all cells. Bottles are placed on a rotatingplatform for 10 minutes and agitated as fast as possible. The debris ispelleted by centrifugation at 20,000×G for 15 minutes. The supernatantis transferred to 250-ml polycarbonate centrifuge bottles, and 0.5volume of 20% PEG8000/2.5M NaCl solution is added. The bottles areshaken overnight on ice. The bottles are centrifuged at 20,000×G for 15minutes, and the supernatants are discarded into a bleach solution. Awhite precipitate (precipitated virus/PEG) forms in two vertical linesalong the walls of the bottles on either side of the spin mark. Using asterile cell scraper, the precipitate from 2 bottles is resuspended in2.5 ml PBS. The virus solution is placed in 2-ml microcentrifuge tubesand centrifuged at 14,000×G in a microcentrifuge for 10 minutes toremove any additional cell debris. The supernatants from the 2-mlmicrocentrifuge tubes are transferred into a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The volume of thevirus solution is estimated, and 0.55 g/ml of CsCl is added. The CsCl isdissolved, and 1 ml of this solution is weighed. The solution istransferred to polycarbonate, thick-walled, 3.2 ml centrifuge tubes(Beckman) and spun at 348,000×G for 3–4 hours at 25° C. The virus formsa white band. Using wide-bore pipette tips, the virus band is collected.

The virus recovered from the gradient includes a large amount of CsCl,which must be removed before it can be used on cells. Pharmacia PD-10columns prepacked with SEPHADEX G-25M (Pharmacia) are used to desalt thevirus preparation. The column is equilibrated with 20 ml of PBS. Thevirus is loaded and allowed to run into the column. 5 ml of PBS is addedto the column, and fractions of 8–10 drops collected. The opticaldensity of a 1:50 dilution of each fraction is determined at 260 nm on aspectrophotometer, and a clear absorbancce peak is identified. Peakfractions are pooled, and the optical density (OD) of a 1:25 dilution isdetermined. OD is convened into virus concentration using the formula(OD at 260 nm)(25)(1.1×10¹²)=virions/ml.

To store the virus, glycerol is added to the purified virus to a finalconcentration of 15%, mixed gently and stored in aliquots at −80° C.

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada)is followed to measure recombinant virus infectivity. Briefly, two96-well tissue culture plates are seeded with 1×10⁴ 293A cells per wellin MEM containing 2% fetal bovine serum for each recombinant virus to beassayed. After 24 hours, 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100 μl of eachdilution is placed in each of 20 wells. After 5 days at 37° C., wellsare read either positive or negative for CPE, and PFU/ml is calculated.

TCID₅₀ formulation used is as per Quantum Biotechnologies, Inc., above.The titer (T) is determined from a plate where virus used is dilutedfrom 10⁻² to 10⁻¹⁴, and read 5 days after the infection. At eachdilution a ratio (R) of positive wells for CPE per the total number ofwells is determined. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml, where F=1+d(S−0.5), S is the sum of the ratios(R), and d is Log₁₀ of the dilution series (e.g., d=1 for a ten-folddilution series). To convert TCID₅₀/ml to pfu/ml, 0.7 is subtracted fromthe exponent in the calculation for titer (T).

Example 5

The human zvegf4 cDNA was cloned into the EcoRV-AscI sites of pZyTrack(Example 4). Ligation was performed using a commercially available DNAligation and screening kit (FAST-LINK kit; Epicentre Technologies,Madison, Wis.).

Zvegf4 was assayed in an aortic ring outgrowth assay (Nicosia andOttinetti, Laboratory Investigation 63:115, 1990; Villaschi and Nicosia,Am. J. Pathology 143:181 –190, 1993). Thoracic aortas were isolated from1–2 month old Sprague-Dawley male rats and transferred to petri dishescontaining HANK's buffered salt solution. The aortas were flushed withadditional HANK's buffered salt solution to remove blood, andadventitial tissue surrounding the aortas was carefully removed. Cleanedaortas were transferred to petri dishes containing EBM basal media,serum free (Clonetics, San Diego, Calif.). Aortic rings were obtained byslicing approximately 1-mm sections using a scalpel blade. The ends ofthe aortas used to hold them in place were not used. The rings wererinsed in fresh EBM basal media and placed individually in wells of a24-well plate coated with basement membrane matrix (MATRIGEL; BectonDickinson; Franklin Lakes, N.J.). The rings were overlayed with anadditional 50 μl of the matrix solution and placed at 37° C. for 30minutes to allow the matrix to gel. Test samples were diluted in EBMbasal serum-free media supplemented with 100 units/ml penicillin, 100μg/ml streptomycin and HEPES buffer and added at 1 ml/well. Backgroundcontrol was EBM basal serum-free media alone. Basic FGF (R&D Systems,Minneapolis, Minn.) at 20 ng/ml was used as a positive control. Zvegf4adenovirus was added to wells, assuming a cell count of 500,000 cellsand a multiplicity of infection of 5000 particles/cell. A nulladenovirus (designated “zPar”) was used as a control. Samples were addedin a minimum of quadruplets. Rings were incubated for 5–7 days at 37° C.and analyzed for growth. Aortic outgrowth was scored by multiple,blinded observers using 0 as no growth and 4 as maximum growth. Zvegf4adenovirus produced a significant increase in outgrowth, comparable tothe bFGF control.

Zvegf4 adenovirus infection produced a significant increase in theoutgrowth of cells as compared to parental virus control. Cells isolatedfrom the matrix surrounding the aortic ring were identified asfibroblasts or smooth muscle cells (SMC) by staining for alpha smoothmuscle actin (characteristic of SMCs), and vimentin and type I collagen(characteristic of fibroblasts). In contrast, there were no endothelialcells detected as indicated by the absence of staining for vonWillebrand's factor, a specific endothelial marker.

Potent induction of cellular outgrowth, similar to that induced bypurified PDGF-AA and PDGF-BB, was also observed following treatment withpurified growth factor domain (mature) zvegf4. These patterns ofoutgrowth were unlike that seen following VEGF treatment, which producedsparser endothelial sprouts. The ability of zvegf4 to induce a responsesimilar to that of PDGF-AA and PDGF-BB, that is a smooth muscle andfibroblast migratory and cyto-kinetic response, correlated with theinvolvement of PDGF receptor stimulation in fibroproliferative responsesof the vasculature.

Example 6

Polyclonal anti-peptide antibodies were prepared by immunizing 2 femaleNew Zealand white rabbits with the peptides huzvegf4-1 (SEQ ID NO:9),huzvegf4-2 (SEQ ID NO:10), or huzvegf4-3 (SEQ ID NO:11). The peptideswere synthesized using an Applied Biosystems Model 431A peptidesynthesizer (Applied Biosystems, Inc., Foster City, Calif.) according tothe manufacturer's instructions. The peptides were conjugated to keyholelimpet hemocyanin (KLH) with maleimide activation. The rabbits were eachgiven an initial intraperitoneal (ip) injection of 200 μg of peptide inComplete Freund's Adjuvant followed by booster ip injections of 100 μgpeptide in Incomplete Freund's Adjuvant every three weeks. Seven to tendays after the administration of the second booster injection (3 totalinjections), the animals were bled, and the sera were collected. Theanimals were then boosted and bled every three weeks.

The zvegf4 peptide-specific rabbit sera were characterized by an ELISAtiter check using 1 μg/ml of the peptide used to make the antibody as anantibody target. The 2 rabbit sera to the huzvegf4-1 peptide had titerto their specific peptide at a dilution of 1:5,000,000. The 2 rabbitsera to the huzvegf4-2 peptide had titer to their specific peptide at adilution of 1:5,000,000. The 2 rabbit sera to the huzvegf4-3 peptide hadtiter to their specific peptide at a dilution of 1:500,000.

The zvegf4 peptide-specific polyclonal antibodies were affinity purifiedfrom the sera using CNBr-SEPHAROSE 4B protein columns (Pharmacia LKB)that were prepared using 10 mg of the specific peptide per gramCNBr-SEPHAROSE, followed by 20× dialysis in PBS overnight.Zvegf4-specific antibodies were characterized by an ELISA titer checkusing 1 μg/ml of the appropriate peptide antigens as antibody targets.The lower limit of detection (LLD) of the anti-huzvegf4-1 affinitypurified antibody on its specific antigen (huzvegf4-1 peptide) was adilution of 0.1 pg/ml. The LLD of the anti-huzvegf4-2 affinity purifiedantibody on its specific antigen (huzveg4-2 peptide) was a dilution of 5ng/ml. The LLD of the rabbit anti-huzvegf4-3 affinity purified antibodyon its specific antigen (huzvegf4-3 peptide) was a dilution of 5 ng/ml.

Example 7

Recombinant amino-terminally Glu-Glu-tagged zvegf4 growth factor domainwith an amino-terminal Glu-Glu (EYMPME; SEQ ID NO:12) tag(zvegf4-nee-GFD) was produced from recombinant baculovirus-infectedinsect cells. 28-liter cultures were harvested, and the media werefiltered using a 0.45 μm filter. Protein was purified from theconditioned media by a combination of cation-exchange chromatography,antibody affinity chromatography, and size-exclusion chromatography.Cultured medium (pH 7.0, conductivity 9 mS) was directly loaded onto a25-ml cation exchange column (POROS 50 HS; PerSeptive Biosystems,Framingham, Mass.). The column was washed with ten column volumes (cv)of PBS, and the bound protein was eluted with a gradient of 20–100% of750 mM NaCl in PBS (Buffer B) for 15 cv followed by 5 cv of 100% BufferB at 5 ml/min. Five-ml fractions were collected. Samples from the columnwere analyzed by SDS-PAGE with silver staining and western blotting forthe presence of zvegf4-nee-GFD. Zvefg4-nee-GFD-containing fractions werepooled and loaded onto an 8-ml anti-Glu-Glu antibody column and elutedwith 50 ml of 0.5 mg/ml EYMPTD (SEQ ID NO:13) peptide (obtained fromPrinceton Biomolecules Corporation, Langhorne, Pa.) in PBS. One-mlfractions were pooled and concentrated to 4 ml using using a BIOMAX-5concentrator (Millipore Corp., Bedford, Mass.) and loaded onto a 16×1000mm gel filtration column (SEPHACRYL S-100 HR; Amersham PharmaciaBiotech, Piscataway, N.J.) at 1.5 ml/minute. Five-ml fractionscontaining purified zvegf4-nee-GFD were pooled, filtered through a 0.2μm filter, aliquoted into 100 μl aliquots, and frozen at −80° C. Theconcentration of the final purified protein was determined by BCA assay(Pierce Chemical Co., Rockford, Ill.) to be 0.4 mg/ml, and the yield wascalculated to be 8.4 mg.

Recombinant zvegf4-nee-GFD was analyzed by SDS-PAGE (NUPAGE 4–12% gel;Novex, San Diego, Calif.) with silver staining (FASTSILVER, GenoTechnology, Inc., Maplewood, Mo.) and Western blotting using antibodiesto the peptide tag. Conditioned media or purified protein waselectrophoresed using an electrophoresis mini-cell (XCELL II mini-cell;Novex) and transferred to nitrocellulose (0.2 μm; Novex) at roomtemperature using a blot module (XCELL II Novex) with stirring accordingto directions provided in the instrument manual. The transfer was run at500 mA for one hour in a buffer containing 25 mM Tris base, 200 mMglycine, and 20% methanol. The filters were then blocked with 10%non-fat dry milk in PBS for 10 minutes at room temperature. Thenitrocellulose was quickly rinsed, then the mouse anti-peptide primaryantibody was added, diluted 1:1000 in PBS containing 2.5% non-fat drymilk. The blots were incubated for two hours at room temperature orovernight at 4° C. with gentle shaking. Following the incubation, blotswere washed three times for 10 minutes each in PBS, then labeled with asecondary antibody (goat anti-mouse IgG conjugated to horseradishperoxidase) diluted 1:1000 in PBS containing 2.5% non-fat dry milk, andthe blots were incubated for two hours at room temperature with gentleshaking. The blots were then washed three times, 10 minutes each, inPBS, then quickly rinsed with H₂O. The blots were developed usingcommercially available chemiluminescent substrate reagents (SUPERSIGNALULTRA reagents 1 and 2 mixed 1:1; reagents obtained from Pierce ChemicalCo.), and the signal was captured using image analysis software(LUMI-IMAGER Lumi Analyst 3.0; Boehringer Mannheim GmbH, Germany) fortimes ranging from 10 seconds to 5 minutes or as necessary.

The purified zvefg4-nee-GFD appeared as two bands on the silver-stainedgel at about 31 and 17 kDa under non-reducing conditions and as a singleband of 17 kDa under reducing conditions. This suggested existence of adimeric form of zvegf4-nee-GFD under non-reducing conditions. Thepurified protein consisted of approximately 90% dimer and 10% monomer.

Example 8

Recombinant human zvegf4 (expressed from the full-length codingsequence) was analyzed for mitogenic activity on rat liver stellatecells (Greenwel et al., Laboratory Invest. 65:644, 1991; Greenwel etal., Laboratory Invest. 69:210, 1993), human aortic smooth muscle cells(Clonetics Corp., Walkersville, Md.), human retinal pericytes (CloneticsCorp.) and human hepatic fibroblasts (Clonetics Corp.). Test samplesconsisted of conditioned media (CM) from adenovirally infected HaCaThuman keratinocyte cells (Boukamp et al., J. Cell. Biol. 106:761–771,1988; Skobe and Fusenig, Proc. Natl. Acad. Sci. USA 95:1050–1055, 1998;obtained from Dr. Norbert E. Fusenig, Deutsches Krebsforschungszentrum,Heidelberg, Germany) expressing full length zvegf4. Control CM wasgenerated from HaCaT cells infected with a parental GFP-expressingadenovirus (zPar). The CM were concentrated 10-fold using a 15-mlcentrifugal filter device with a 10K membrane filter (ULTRAFREE;Millipore Corp., Bedford, Mass.), then diluted back to 1× with ITSmedium (serum-free DMEM/Ham's F-12 medium containing 5 μg/ml insulin, 20μg/ml transferrin, and 16 pg/ml selenium). Cells were plated at adensity of 2,000 cells/well in 96-well culture plates and grown forapproximately 72 hours in DMEM containing 10% fetal calf serum at 37° C.Cells were quiesced by incubating them for approximately 20 hours in ITSmedium. At the time of the assay, the medium was removed, and testsamples were added to the wells in triplicate. For measurement of[³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, media were removed and cells were incubatedwith 0.1 ml of trypsin until cells detached. Cells were harvested onto96-well filter plates using a sample harvester (FILTERMATE harvester;Packard Instrument Co., Meriden, Conn.). The plates were then dried at65° C. for 15 minutes, sealed after adding 40 μl/well scintillationcocktail (MICROSCINT O; Packard Instrument Co.) and counted on amicroplate scintillation counter (TOPCOUNT; Packard Instrument Co.).Results, presented in Table 2, demonstrated that zvegf4 CM hadapproximately 7-fold higher mitogenic activity than control CM onpericytes cells and approximately a 1.5–2.4-fold higher mitogenicactivity on the other cell types tested. While not wishing to be boundby theory, it is believed that the observed activity may be due to thepresence of cleaved zvegf4 protein (i.e., growth factor domain).

TABLE 2 CPM incorporated Zvegf4 (1 × CM) zPar (1 × CM) Sample Mean St.dev. Mean St. dev. Human retinal pericytes 3621 223 523 306 Humanhepatic fibroblasts 7757 753 3232 264 Human aortic SMC 2009 37 1263 51Rat liver stellate cells 34707 1411 14413 1939

Example 9

Recombinant, C-terminally glu-glu tagged, human zvegf4 (expressed inbaculovirus-infected cells expressing a full-length zvegf4 codingsequence) was analyzed for mitogenic activity on human aortic smoothmuscle cells (HAoSMC) (Clonetics), human retinal pericytes (Clonetics)and human aortic adventitial fibroblasts (AoAF) (Clonetics). Cells wereplated at a density of 2,000 cells/well in 96-well culture plates andgrown for approximately 72 hours in DMEM containing 10% fetal calf serumat 37° C. Cells were quiesced by incubating them for 20 hours in ITSmedium. At the time of the assay, the medium was removed, and testsamples were added to the wells in triplicate. Purified protein in abuffer containing 0.1 % BSA was serially diluted into ITS medium atconcentrations of 1 μg/ml to 1 ng/ml and added to the test plate. Acontrol buffer of 0.1% BSA was diluted identically to the highestconcentration of zvegf4 protein and added to the plate. For measurementof [³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, mitogenic activity was assessed by measuringthe uptake of [³H]thymidine. Media were removed, and cells wereincubated with 0.1 ml of trypsin until cells detached. Cells wereharvested onto 96-well filter plates using a sample harvester(FILTERMATE harvester; Packard Instrument Co., Meriden, Conn.). Theplates were then dried at 65° C. for 15 minutes, sealed after adding 40μl/well scintillation cocktail (MICROSCINT O; Packard Instrument Co.)and counted on a microplate scintillation counter (TOPCOUNT; PackardInstrument Co.). Results, presented in Table 3 demonstrated that 80ng/ml zvegf4 had approximately 1.7-fold higher mitogenic activity onpericytes, 3.2-fold higher activity on aortic SMCs, and 2.6-fold higheractivity on aortic fibroblasts as compared to the buffer control.

TABLE 3 CPM Incorporated Pericytes HAoSMC AoAF Sample Mean St. dev. MeanSt. dev. Mean St. dev. Zvegf4, 80 96.7 18.2 488.7 29.6 177.0 1.0 ng/mlZvegf4, 20 81.7 11.7 211.7 50.8 107.7 20.1 ng/ml Zvegf4, 5 67.3 6.7191.7 4.5 123.7 10.5 ng/ml Buffer control 58.7 8.5 152.3 40.1 68.7 8.3

Example 10

The protein-coding region of human zvegf4 DNA was amplified by PCR usingprimers that added PmeI and AscI restriction sites at the 5′ and 3′termini, respectively. The resulting zvegf4 cDNA was cloned into theEcoRV-AscI sites of pZyTrack (Example 4). Recombinant adenovirus wasgenerated in 293A cells and purified on CsCl gradients. Viral particlenumbers were determined by spectrophotometry, and infectious particlenumbers were determined by TCID₅₀ assay. The virus was designatedAdZyvegf4.

Eight-week-old C57BL/6 mice were infected with AdZyvegf4 to determinethe effects on serum chemistry, complete blood counts (CBC), body andorgan weight changes, and histology. On day −1, the mice were tagged,individually weighed, and group normalized for separation into treatmentgroups (4 mice per cage). Group 1 mice (n=8 females, 7 males) receivedGFP (control) adenovirus, 1×10¹¹ particles. Group 2 mice (n=8 females, 6males) received zvegf4 adenovirus, 1×10¹¹ particles. Group 3 mice (n=8females, 8 males) were untreated controls. On day 0, the mice receivedinjections of the appropriate adenovirus solution. On day 10, blood wascollected (under ether anesthesia) for CBCs and clinical chemistrymeasurements. On day 20, mice were weighed and sacrificed by cervicaldislocation after collecting blood (under ether anesthesia) for CBCs andclinical chemistry measurements. Selected tissues were fixed andevaluated for morphological changes. The following pathological findingswere noted in the majority (80–100%) of the animals treated with theAdZyvegf4 adenovirus, and were not observed in either of the other twogroups.

In the liver, there was moderate proliferation of sinusoidal cells,especially cells with small ovoid nuclei and no observable cytoplasmlining the sinusoids that were more clustered in the venous regions ofthe hepatic lobule. The cells appeared to be spindle Ito (or stellate)cells, which are a major cell type incriminated in the onset andprogression of hepatic fibrosis.

In all AdZyvegf4-treated animals, the glomeruli of the kidneys wereenlarged and were characterized by increased cellularity, diagnosed asproliferative glomerulopathy. Because of their location andmorphological characteristics, the proliferating cells within theglomerulus that contributed to its enlargement were most likelymesangial cells. In addition, there was evidence of tubular regenerationin many of the kidneys, characterized by tubular epithelial cells withincreased basophilia.

An increased amount of bronchoalveolar lymphoid tissue was noted in thelungs of the AdZyvegf4-treated animals. Bronchoalveolar lymphoid tissueconsisted predominantly of clusters of lymphocytes admixed with fewernumbers of plasma cells around vessels within the lung parenchyma, asign of lung inflammatory response, which is an important initiator andparticipant in several forms of lung fibrosis.

In the femur, the majority of animals displayed minimal to severeendosteal bone filling the marrow space, with decreased amounts ofhematopoietic elements resulting from loss of marrow space due to theproliferating endosteal bone. In addition, four of six male and two ofeight female animals had proliferation of stromal cells, which wascharacterized by an increased number of spindle-shaped cells.

Example 11

90 μg of full-length, recombinant human zvegf4 protein was dissolved in500 μl PBS containing 2 mCi Na¹²⁵I (Amersham Corp.). One derivatized,nonporous polystyrene bead (IODO-BEADS; Pierce, Rockford, Ill.) wasadded, and the reaction mixture was incubated one minute on ice. Theiodinated protein was separated from unincorporated ¹²⁵I by gelfiltration using an elution buffer of 10% acetic acid, 150 mM NaCl, and0.25% gelatin. The active fraction contained 29 μg/ml ¹²⁵I-zvegf4 with aspecific activity of 3.0×10⁴ cpm/ng.

The following cell times were plated into the wells of a 24-well tissueculture dish and cultured in growth medium for three days:

-   -   1. Human retinal pericytes, passage 6 (pericytes).    -   2. Rat stellate cells, passage 8.    -   3. Human umbilical vein endothelial cells, passage 4 (HUVEC).    -   4. Human aortic adventitial fibroblasts, passage 5 (AoAF).    -   5. Human aortic smooth muscle cells, passage 2 (AoSMC).        Cells were washed once with ice-cold binding buffer (HAM'S F-12        containing 2.5 mg/ml BSA, 20 mM HEPES, pH 7.2), then 250 μl of        the following solutions was added to each of three wells of the        culture dishes containing the test cells. Binding solutions were        prepared in 5 mL of binding buffer with 250 pM ¹²⁵I-zvegf4 and:    -   1. No addition.    -   2. 25 nM zvegf4.    -   3. 25 nM zvegf3 (PDGF-C).    -   4. 25 nM PDGF-AA.    -   5. 25 nM PDGF-BB.        The reaction mixtures were incubated on ice for 2 hours, then        washed three times with one ml of ice-cold binding buffer. The        bound ¹²⁵I-zvegf4 was quantitated by gamma counting a        t-octylphenoxypolyethoxyethanol (TRITON X-100) extract of the        cells.

Results, shown in Table 4, indicate binding of zvegf4 to pericytes,stellate cells, AoAF, and AoSMC, but not to HUVEC. The first columnrepresents total CPM ¹²⁵I-zvegf4 bound/well. The second column is¹²⁵I-zvegf4 bound/well when blocked with cold ligand. The differencebetween the two numbers represents specific binding.

TABLE 4 ¹²⁵I-zvegf4 Bound w/cold Cell Type ¹²⁵I-zvegf4 Bound (CPM)zvegf4 (CPM) 1. Pericytes 3083 +/− 864 623 +/− 60 2. Stellate Cells 2131+/− 450  413 +/− 164 3. HUVEC 485 +/− 91 227 +/− 13 4. AoAF 1544 +/− 131300 +/− 15 5. AoSMC 1628 +/− 203 440 +/− 46

Zvegf4 binding was not significantly reduced by PDGF-AA of PDGF-BB (datanot shown). These results indicate that full-length zvegf4 can bind tothe tested cells at binding sites distinct from those for the AA and BBisoforms of PDGF. These zvegf4 binding sites may be either (1) sites onknown PDGF receptors that are distinct from the binding sites for the AAand BB isoforms, or (2) one or more different molecules, such ascell-surface semaphorins.

Example 12

The activity of recombinant human zvegf4 growth factor domain was testedon BHK cell lines that stably expressed the α subunit of the PDGFreceptor, the β subunit of the PDGF receptor, or both the α and βsubunits of the PDGF receptor. Wild-type BHK 570 cells (which do notrespond to PDGF because they do not express adequate levels of PDGFreceptors) and stable BHK cell lines that expressed the human PDGFreceptor subunits were plated at 10–15×10³ cells/well in 96-well cellculture trays in DMEM (Life Technologies, Gaithersburg, Md.)supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc.,Logan, Utah). The medium of the stable clones was further supplementedwith 200 nM methotrexate to maintain stable selection. At 70–80%confluence (the next day), the growth medium was replaced withserum-free medium, and the cells were infected with an adenoviralreporter construct (designated KZ 136) containing a firefly luciferasegene under the control of an SRE-STAT promoter at 1,000:1 multiplicityof infection (1,000 viral particles per cell.). Twenty-four hours later,the medium was once more replaced with serum-free medium, andrecombinant human zvegf4 growth factor domain was added to the cells.Four hours later, the cells were lysed, and the luciferase activity inthe lysate was determined using a commercially available kit (obtainedfrom Promega Corporation, Madison, Wis.) and a luminometer device(LUMINOSKAN; Labsystems Oy, Helsinki, FI) to detect the emittedluminescence. As shown in Tables 5–7, zvegf4 triggered responsed in allthree receptor-expressing BHK cell lines (but not in wild-type BHKcells, not shown), indicating that is can signal through αα, ββ and αβPDGF receptor complexes.

TABLE 5 BHK expressing the PDGF receptor alpha subunit Zvegf4 Luciferase(ng/ml) Units 0 2.14 0.3 2.12 1 2.92 3 4.50 10 9.60 30 13.30 100 21.95

TABLE 6 BHK expressing PDGF receptor beta subunit Zvegf4 Luciferase(ng/ml) Units 0 4.49 0.3 8.53 1 14.77 3 20.91 10 37.22 30 40.16 10036.41

TABLE 7 BHK cells expressing PDGF receptor alpha and beta subunitsZvegf4 Luciferase (ng/ml) Units 0 6.64 0.3 13.99 1 19.11 3 26.21 1044.25 30 61.15 100 60.25

Example 13

Human aortic smooth muscle cells at passage 6 (Clonetics) were plated at10×10³ cells/well in 96-well cell culture trays in DMEM supplementedwith 10% fetal bovine serum. At confluence (the next day), the growthmedium was replaced with serum-free DMEM containing 0.1% BSA, and thecells were returned to the incubator, allowing for partial growtharrest. Twenty-four hours later, the media were once more replaced withserum-free medium with BSA. Recombinant human zvegf4 growth factordomain (30 ng/ml final concentration in the well) was mixed withneutralizing monoclonal antibodies against the alpha or beta PDGFreceptor subunits or with non-immune mouse IgG (20 μg/ml finalconcentration in the well) for 10 minutes at room temperature. Themixture was then added to the cells. [³H]-thymidine at 1 μCi/ml(Amersham, final concentration in the well) was added immediatelyafterwards, and the cells were returned to the incubator for anadditional 24 hours to allow for [³H]-thymidine incorporation into newlysynthesized DNA. The cells were washed twice to remove unincorporatedlabel and were harvested using a sample harvester (FILTERMATE 196harvester; Packard Instrument Co.). Incorporated thymidine wasdetermined using a scintillation counter (TOPCOUNT; Packard InstrumentCo.). Results from triplicate well determinations, expressed asmean±standard deviation of cpm of radioactivity per well, are shown inTable 8. “Response” indicates the fold increase in thymidineincorporation resulting from the addition of zvegf4. The data show thatthe response of human aortic smooth muscle cells to zvegf4 wassubstantially reduced by both anti-alpha and anti-beta PDGF receptorsubunit neutralizing antibodies, indicating that both PDGF receptorsubunits are bound by zvegf4.

TABLE 8 [³H]-Thymidine Antibody zvegf4 (cm) Response Non-immune serum(Control) −  81 ± 21 Non-immune serum (Control) + 696 ± 60   8-foldanti-PDGF-Rβ − 116 ± 19 anti-PDGF-Rβ + 151 ± 15 1.4-fold anti-PDGF-Rα −102 ± 32 anti-PDGF-Rα + 322 ± 64 3.2-fold

Example 14

Rat stellate cells were grown in 48-well tissue clusters (FALCON; BDLabware, Bedford, Mass.) in DMEM (Life Technologies, Inc.) supplementedwith 10% fetal bovine serum (Hyclone Laboratories, Inc.). At 80%confluence, the cells were switched to growth-arrest medium bysubstituting 0.1% BSA (Sigma-Aldrich Corp., St. Louis, Mo.) for serum.Two days later the growth-arrest medium was replaced with the samemedium, and recombinant human zvegf4 growth factor domain was added tothe cells. After 48 hours, the conditioned media were collected, andTGF-β1 levels were determined using an ELISA kit (obtained from R&DSystems, Minneapolis, Minn.). Results are shown in Table 9.

TABLE 9 Treatment pg TGF-β1 per well BSA Control  2 ± 3 Zvegf4 3 ng/ml 32 ± 28 Zvegf4 30 ng/ml 120 ± 12 Zvegf4 300 ng/ml 175 ± 71

Example 15

OC10B mouse osteoblasts (Thomson et al., J. Bone Min. Res.,13(2):195–204, 1998) were grown in 96-well tissue clusters (FALCON)until confluence in DMEM (Life Technologies, Inc.) supplemented with 10%fetal bovine serum (Hyclone Laboratories, Inc.). They were then switchedto growth-arrest medium by substituting 0.1% BSA (Sigma-Aldrich Corp.)for serum. Forty-eight hours later, the growth-arrest medium wasreplaced with the same medium, and recombinant human zvegf4 growthfactor domain was added to the cells. The cells were pulsed with 1μCi/ml [³H]-thymidine (NEN Life Science Products, Inc., Boston, Mass.)for 8 hours, 16–24 hours after addition of zvegf4. The radioactivityincorporated by the cells was determined by harvesting the cells with asample harvester and counting the incorporated label using a microplatescintillation counter.

Results, shown in Table 10, indicate that zvegf4 directly stimulatesosteoblast proliferation. Zvegf4 antagonists may therefore be useful inreducing growth of osteoblasts, such as in osteosarcomas or osteoblasticprostate metastases.

TABLE 10 Treatment cpm/well (10⁻³) BSA Control 17 ± 2  Zvegf4 1 ng/ml 36± 11 Zvegf4 3 ng/ml 42 ± 8  Zvegf4 10 ng/ml 51 ± 17 Zvegf4 30 ng/ml 59 ±11 Zvegf4 100 ng/ml 51 ± 25 Zvegf4 300 ng/ml 63 ± 13

Example 16

OC10B cells in vitro can differentiate into both osteoblasts andadipocytes (fat cells) when grown in a medium containing 100 μg/mlascorbic acid and 10 mM beta-glycerophosphate (Thomson et al., ibid.).This differentiation recapitulates the in vivo physiological processwhereby both cell lineages are derived from a common, bi-potentialprogenitor.

OC10B cells were cultured in the presence of ascorbic acid andbeta-glycerophosphate for 10 days. Zvegf4 suppressed differentiation ofthe cells into adipocytes, as determined by the absence of cellscontaining light-reflective fat droplets. In contrast, there was anincrease in the number and size of mineralized foci as assessed by VonKossa staining.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of reducing kidney fibrosis in a mammal comprisingadministering to a mammal having a fibroproliferative disorder of kidneya composition comprising a humanized monoclonal antibody or a humanmonoclonal antibody that specifically binds to an epitope of a proteinas shown in SEQ ID NO:2 from amino acid residue 19 to amino acid residue370, in combination with a pharmaceutically acceptable delivery vehicle,wherein said antibody is an antagonist of said protein, in an amountsufficient to reduce kidney fibrosis in the mammal, wherein thefibroproliferative disorder is characterized by mesangial cellproliferation.
 2. The method of claim 1 wherein the antibody is ahumanized monoclonal antibody.
 3. The method of claim 1 wherein thefibroproliferative disorder of kidney is membranoproliferativeglomerulonephritis, diffuse proliferative glomerulonephritis, diabeticnephiopathy, or lupus nephritis.
 4. The method of claim 1 wherein theantibody binds to an epitope of a protein as shown in SEQ ID NO:2 fromamino acid residue 258 to amino acid residue
 370. 5. The method of claim4 wherein the fibroproliferative disorder of kidney ismembranoproliferative glomerulonephritis, diffuse proliferativeglomerulonephritis, diabetic nephropathy, or lupus nephritis.
 6. Themethod of claim 1 wherein the fibroproliferative disorder of kidney isfurther characterized by extracellular matrix accumulation.